Attempts have been made over the past years to use antibodies in combination with agents that directly exert a toxic effect on target cells (cytotoxic agents, cytotoxins) in order to provide a selective action on target cells and to prevent and minimize the non-specific effect on other cells. The combinations suggested have ranged from covalently bonded complexes using linkage-providing molecules and non-covalently bonded complexes to simple mixings (e.g. Ghose et al., J. Natl. Cancer Inst. 61(1979)657-676 and Carlsson et al., Biotechnology 7(1989)567-73). Suggested cytotoxins have been i.a. diphteria toxin, ricin, subunit A of ricin, gelonin, and Pseudomonas aeruginosa exotoxin A (Takeda Chemical Ind., EP-A-336,405 and Pastan et al., WO-A-88/00703, both of which have been cited in connection with the priority application, SE-9002479).
With the advent of the hybridoma technology and the accompanying availability of monoclonal antibodies, it has been feasible to use the concept of complexes between antibodies and cytotoxic agents to more specifically direct the cytotoxic agents to the intended target cell population.
In view of the recognized damaging effect of cytotoxic agents on other cells than target cells, one has suggested to replace the cytotoxic agents with immune stimulators that trigger T-lymphocytes and activate CTLS. Specific proposals have been concerned with antibodies conjugated to
(i) antibodies that are directed against a T-cell receptor or compounds that are able to bind to a T-cell receptor (Mass. Inst. Techn., EP-A1-180,171); PA1 (ii) compounds, such as antigens, mitogens, other foreign proteins, and peptides that activate cytotoxic T-cells (Neorex Corp., EP-A1-334,300); PA1 (iii) MHC antigens, (Behringwerke AG, EP-A1-352,761); PA1 (iv) antigens against which the individual to be treated has immunity, (Med. Res. Counc. WO-A-90/11779 (publ. 1990-10-18)); and PA1 (v) an unnamed bacterial enterotoxin (Ochi and Wake, UCLA-symposium: Cellular Immunity and the Immunotherapy of Cancers, Jan. 27-February 3, 1990, Abstract CE 515. page 109). PA1 (i) comprising (1) an antibody directed against target cells, and (2) a superantigen, i.e. a structure that is recognized (interact with or bind to) and activate T-cells, in particular CTLs; PA1 (ii) methods for destroying target cells, in particular in connection with therapeutical treatment methods contemplated on mammals, and for specific activation of T-cells, such as CTLS; PA1 (iii) method of synthesis for the conjugates; and PA1 (iv) pharmaceutical compositions containing the conjugates and preparation methods for the compositions.
However, the immune stimulators suggested hitherto have been either too specific or too general in their action. For instance classical antigens activate only about 1 out of 10.sup.5 T-cells while mitogens are potentially capable of activating a majority of the T-cells.
It has been recognized that certain-agents mediate activation of a-moderate ratio of T-cells; i.e. they activate T-cells at a relatively high frequency, but far from 100% (Fleischer et al., J. Exp. Med. 167(1988)1697-1707; and White et al., Cell 56(1989)27-35, both articles being incorporated by reference). This type of agents are more effective activators than classical antigens and they accordingly have been named superantigens (for a review see Kappler and Marrack, Science 248:705, (1990)). It has further been demonstrated (Dohlsten et al., Immunol. 71(1990)96-100; and Hedlund et al., Cell. Immunol 129(1990)426-34, both articles being incorporated by reference) that the superantigens known so far have the capacity to bind to MHC Class II molecules on target cells and activate cytotoxic T-cells bearing the proper T-cell receptor V beta chain. The published data indicate that the MHC binding is a prerequisite for T-cell binding and activation to occur. It can not be excluded that in the future superantigens will be found that act through a T-cell receptor V alpha chain or other surface structures only found on subpopulations of T-cells.
The immunomodulatory effect of the superantigen Staphylococcus enterotoxin A (SEA) has also been described by Platsoucas et al (Cell Immunol. 97(1986)371-85).
Most of the presently known superantigens have earlier been recognized as toxins and all of them have been of microbial origin. Staphylococcal enterotoxins for instance are enterotoxic and activate T-cells, and the two effects are discernible from each other (Fleischer et al., Cell. Immunol. 118(1989)92-101; Alber et al., J. Immunol 144(1990)4501-06; and Infect. Immun. 59(1991)2126-34).
It has previously been suggested to use superantigens in order to direct CTL mediated lysis of cells carrying MHC Class II antigens (Pharmacia AB, WO-A-91/04053, publ. 1991-04-04). Wo-A-91/04053 covers, but does not explicitly mention, superantigens that are incorporated into covalent immunoconjugates.
Cells lacking MHC Class II or expressing marginal amounts of MHC Class II proteins do, however, not bind sufficient amounts of superantigens in order to efficiently direct lysis of them by CTLS. Thus due to the general abundance of cells carrying MHC Class II antigens and the non-abundance of MHC Class II antigens on most tumour cells, superantigens should be of low value for the specific killing of such unwanted cells.
However, we have found that a specific cell-killing effect mediated by CTLs can be achieved with superantigens, if they are covalently linked to an antibody directed against an epitope that is specific for the cell to be killed. The activation of the immune system may induce target cells lacking MHC Class II antigens to express them, which may potentiate the desired lytic effect.